Purdue University researchers have confirmed the long-held hypothesis that sorghum deters insects from feeding on its leaves by releasing hydrogen cyanide.

Mitch Tuinstra and Brian Dilkes found that insects preferred the leaves of a mutant sorghum plant with an abnormally slow release of cyanide to those of a wild-type sorghum plant with a normal cyanide-release rate. Fall army worms fed on the leaves of the mutant sorghum even though the leaves contained similar levels of dhurrin - the chemical compound responsible for synthesizing hydrogen cyanide - as those in normal sorghum plants.

"This study separates for the first time the accumulation of dhurrin from the release of hydrogen cyanide," said Dilkes, assistant professor of horticulture and landscape architecture. "Both the mutant and normal sorghum plants contain dhurrin, but it's the rate of cyanide release that causes the insects to avoid one in favor of the other. It's a beautiful interaction between animal behavior and plant chemistry."

Sorghum bicolor , the cultivated species of sorghum, is a critically important cereal grass used for food and animal forage in many parts of the developing world and is a promising bioenergy crop in the U.S. Its ability to thrive in arid environments makes it a more water-efficient crop than corn.

While the grain of sorghum is edible, its leaves can sometimes contain levels of hydrogen cyanide that are toxic to humans and animals. Livestock producers have long known that feeding sorghum leaves harvested at certain growth stages, and particularly under stress conditions such as drought, can result in cyanide poisoning of livestock. When properly managed, however, sorghum leaves can be safe forage for cattle.

Identification of genes that control cyanide production and release could lead to the development of cyanide-free sorghum plants.

Tuinstra and Dilkes identified a sorghum mutant with an exceptionally slow cyanide-release rate. They located the gene responsible for the defect by using next-generation sequencing, a technique that randomly generates short sequences from a genome - the total genetic content of an organism - and stitches them back together. Next-generation sequencing works like a text editor, said Tuinstra, professor of plant breeding and genetics.

"It's just like taking a 10-word sentence from a book and asking where it belongs," he said. "It finds the location of a specific sequence inside the species genome. The mutation is like a misplaced period in the middle of a sentence - it signals the reader to stop. In the case of the sorghum mutant, it halts the production of a functional protein."

The sequencing technique allowed Tuinstra and Dilkes to identify the single nucleotide within the sorghum genome of 790 million base pairs that slowed the release of cyanide in the mutant plant.

"This study is an example of how new methods in DNA sequencing can now be used to unlock the genetic mechanisms of sorghum performance," Tuinstra said.

After cloning the mutant, the researchers tested insect feeding preference by releasing fall army worms onto mutant and normal sorghum plants. Though both types of sorghum contained normal levels of dhurrin, the insects avoided the normal sorghum plants, settling and feeding on the leaves of the mutant sorghum instead. While the mutant contains the compounds necessary to generate cyanide, it cannot release cyanide quickly enough to ward off pests, Tuinstra said.

Next-generation sequencing is more often used in plant species with genomes much smaller than sorghum. The study clears the way to use advanced sequencing techniques to identify genes and gene functions in plants with large genomes, Dilkes said.

"We've demonstrated that these sequencing tools are robust enough to apply to organisms with complex genomes," he said. "If we can use them in sorghum, we can use them in other crops. In terms of identifying genes of interest in complex organisms, we're open for business."

The research was conducted using funds from the U.S. Department of Energy, the International Sorghum and Millet Collaboration Research Support Program and an Agriculture and Food Research Initiative Competitive Grant.